Effects of Radiation on Metabolic activities of Aureobasidium melanogenum based on Biolog FF system jing zhu ( [email protected] ) Xinjiang Academy of Agricultural Sciences zhi dong Zhang Xinjiang Academy of Agricultural Sciences qi yong Tang Xinjiang Academy of Agricultural Sciences mei ying Gu Xinjiang Academy of Agricultural Sciences li juan Zhang Xinjiang Academy of Agricultural Sciences wei Wang Xinjiang Academy of Agricultural Sciences Research article Keywords: Biolog FF system, Aureobasidium melanogenum, Radiation, Metabolic activity Posted Date: September 3rd, 2020 DOI: https://doi.org/10.21203/rs.3.rs-65430/v1 License: This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/16 Abstract Background Under the radioactive stress, fungi show extremely strong radiation tolerance and highly diverse genomic structure, as well as abnormal character and nutritional requirements. The genus Aureobasidium can tolerate various adversities such as ultraviolet rays, hyperosmotic stress and heavy metal poisoning. Results The experiment used 60Co γ radiation, and set four radiation doses of 0 Gy, 2,500 Gy, 5,000 Gy, and 10,000 Gy. Biolog FF technology was used to detect the utilization of carbon source in FF microplate by Aureobasidium melanogenum F119, and to analyze the effects of the radiation on carbon metabolic activity of A. melanogenum F119. There were signicant differences in AWCD (Average well color development) values after explored with different radiation doses. With the increase of radiation dose, the metabolic activity of cells decreased signicantly. The utilization of carboxylic acids and amino acids showed a downward trend, while that of carbohydrates showed an upward trend. At the same time, as the radiation dose increased, the utilization of carbon source changed. Among the 95 carbon sources in the Biolog FF microplates, a total of 46 carbon sources were used, and 7 carbon sources increased in average utilization rate with the increase of radiation dose. Among them, the utilization of D-ribose, sucrose, D-arabinose and L-arabinose is the most obvious. Conclusion Radiation can obviously reduce the metabolic activity of A. melanogenum F119, and the utilization of carbon source will change signicantly under high doses of radiation, which may be an effective mechanism for its adaptation to radiation. Background Ionizing radiation (IR) can act on biological macromolecules, produce radiochemical effects and damage to organisms, such as ionization, free radicals production, nucleic acid damage, etc., [1], which is a deadly environmental stress. However, organisms have produced various survival strategies to deal with radiation during evolution, which can minimize the damage of radiation to the basic metabolic mechanism of organisms and repair them. In the study about microorganisms in the radiation zone, fungi were found to be an important organism enriched in radioactive materials [2–4]. Under radioactive stress, fungi show extremely strong radiation tolerance and highly diverse genomic structure [5], as well as special appearance and nutritional requirements [4], and produce abundant secondary metabolites such as pigments, enzymes and polysaccharides, etc. The genus Aureobasidium is a kind of the polymorphic fungi with yeast and mycelium morphology. Its chlamydospores accumulate melanin [6] and can resist various adversities such as ultraviolet rays, hyperosmotic stress and heavy metal poisoning [7, 8]. It can also be used for producing single cell protein, polysaccharide, extracellular polysaccharide, pectinase, pigment, etc. [9]. In previous research, our laboratory obtained many strains of genus Aureobasidium [10]. These strains not only have high-dose radiation (60Co γ-ray 20 kGy) tolerance, but also have strong resistance to ultraviolet (UV) radiation and multiple heavy metals. Soluble melanin is also produced in large quantities during the growth of these strains, which can effectively improve the survival rate of microorganisms under UV irradiation. Thus demonstrating that it has good protection to UV radiation [11]. However, there were few reports on the changes of growth, Page 2/16 metabolic activity and the underlying stress mechanism and radiation tolerance mechanism of such strains under radiation stress. Biolog microbial automatic analysis system is a set of microbial carbon source metabolism and identication analysis system. Different identication analysis plates can identify and analyze nearly 2,000 kinds of microorganisms, including bacteria, yeast, lamentous fungi and environmental microbial community diversity [12, 13]. The Biolog FF identication microplates can be used for the identication of lamentous fungi, and also for the analysis of carbon source metabolic diversity of fungal communities in the environment. A black yeast-like fungus F119 was isolated from radiation- contaminated soil samples of Xinjiang. By the colony morphology, mycelial characteristics and phylogenetic tree analysis, it was identied as A. melanogenum F119. Different 60Co γ radiation doses were set up for the radiation experiment. Biolog FF technology was used to detect and analyze the carbon source utilization of the strain after radiation. According to average well color development (AWCD) value of a single well, the effect of radiation on the metabolic activity of A. melanogenum F119 was analyzed. So as to analyze the radiation adaptation mechanism of A. melanogenum F119 and provide theoretical basis for development and application of radiation-resistant fungi. Results Identication of Strain F119 The strain F119 can grow at 4 ℃-30 ℃, the optimal culture temperature is 28 ℃, and resistant to 15% NaCl. the initial colonies were all yeast-like, off-white on Czapek Dox Agar at 28 ℃. In the later period, the fungal mycelium was embedded in the culture medium. After 3 days of culture, the colonies turned dark or blackish-brown, and raised (Fig. 1). After 7 days of cultivation, the colony size was 0.2 cm-1.0 cm; the optimal initial growth pH was 6.0. Microscopic observation showed that the vegetative mycelium was transparent, smooth, thin-walled, and had a septum, which was converted into black mycelium in the middle and late stages of culture (Fig. 2). The conidia were transparent to dark brown and they were single cells, smooth, oval, 4.5–10 × 3.0–10 µm. Comparisons with LSU rDNA D1/D2 domain sequences from GenBank database revealed that strain F119 (557 bp, GenBank accession number JN854147) shared 95.1-99.5% similarity with those of the species of the genus Aureobasidium, and the most closely related strains were A. melanogenum CBS 105.22T and A. melanogenum CBS 621.80T. Based on the analysis of phylogenetic trees, strain F119 merits recognition as a distinct genomic species of the genus Aureobasidium. Metabolic proling of A. melanogenum F119 in the Biolog FF Microplate The metabolic abilities of this isolate were tested by using the Biolog FF Microplate which included 95 different carbon sources. The absorbance values at different times (0-144 h) were obtained to study the abundance index of 95 carbon sources metabolized by the strain. It can be seen from Fig. 4, the strain used the most types of carbon source at 72 h. The strain F119 was able to eciently metabolize 46 carbon sources for growth of the fungus. It included 24 types of carbohydrates: D-arabitol, arbutin, D-cellobiose, D-arabinose, L-arabinose, i-erythritol, D-fructose, gentiobiose, α-D-glucose, maltose, maltotriose, D-mannitol, D-mannose, D-melezitose, β-methyl-D-glucose glycosides, palatinose, D-psicose, D- ranose, L-rhamnose, D-ribose, stachyose, sucrose, D-trehalose, D-xylose; 6 types of amino acids: L-alanine, L- asparagine, L-aspartic acid, L-glutamic acid; L-proline, L-pyroglutamic acid; 8 types of carboxylic acids: D-galacturonic acid, D-glucuronic acid, fumaric acid, y-hydroxybutyric acid, α-ketoglutaric acid, L-malic acid, quinic acid, succinic acid; 1 type of amines: succinamic acid; 3 types of polymers: Tween 80, dextrin, glycogen; 4 types of miscellaneous: salicin, glycerol, bromosuccinic acid, succinic acid mono-methyl ester (Fig. 5). Metabolic proling of A. melanogenum F119 with exposure to radiation Page 3/16 In order to explore the utilization capacity of carbon sources from the strain F119, AWCD value was used to measure it [14]. The collected data was analyzed and the average absorbance value was calculated. The results showed: in the control strain, the AWCD value increased with the increase of the cultivation time, tended to be stable at 96–168 h, and eventually reached the maximum at 144 h. The AWCD values of the strain showed the same trend under different radiation doses. Under 60Co γ-ray 2,500 Gy irradiation, the cell metabolic activity was close to the control group, which were respectively 0.2357 and 0.1628; when the radiation dose was greater than 5,000 Gy, the cell metabolic activity dropped rapidly to 0.0782, lower than 50% of the control group; when the radiation dose reached 10,000 Gy, the cell metabolic activity was 0.0162, only about 6% of the control group. It showed that under low dose radiation, the growth and metabolism of the strain were less affected. However, when the radiation dose was greater than 5,000 Gy, the cell growth rate and metabolic activity decreased signicantly. When the radiation dose reached 10,000 Gy, the cell growth was almost stagnant and the metabolic activity was extremely low (Fig. 6). Utilization of carbon sources of A. melanogenum F119 under different radiation doses According to the change of AWCD value and the utilization of carbon sources, this study selected 72 h results to analyze the utilization percentage of six major carbon sources for each sample. It can be seen from Fig. 7 that obvious differences in the utilization percentage of the six major carbon source of strain. With the increase of radiation dose, the utilization percentage of carbohydrates showed an upward trend, while the utilization percentage of carboxylic acids and amino acids showed a downward trend. The utilization percentage of other types of carbon sources was basically the same.